The accumulation of anthocyanin pigments is an important determinant of fruit quality. Pigments provide essential cultivar differentiation for consumers and are implicated in the health attributes of apple fruit (Boyer and Liu, 2004).
Anthocyanin pigments belong to the diverse group of ubiquitous secondary metabolites, collectively known as flavonoids. In plants, flavonoids are implicated in numerous biological functions, including defence, whilst the pigmented anthocyanin compounds in particular play a vital physiological role as attractants in plant/animal interactions.
The predominant precursors for all flavonoids, including anthocyanins, are malonyl-CoA and p-coumaroyl-CoA. From these precursors the enzyme chalcone synthase (CHS) forms chalcone, the first committed step towards anthocyanin production and the establishment of the C15 backbone. Chalcone is then isomerised by chalcone isomerase (CHI) to produce chalcone naringenin and from there a hydroxylation step via flavanone 3β-hydroxylase (F3H) converts naringenin to dihydroflavonol. Reduction of dihydroflavonon by dihydroflavolon 4-reductase (DFR) produces leucoanthocyanin which is converted into the coloured compound anthocyanindin by leucoanthocyanidin dioxygenase (LDOX) whilst the final glycosylation step is mediated by uridin diphosphate (UDP)-glucose:flavonoid 3-0-glucosyltransferase (UFGT). The difference in anthocyanin colour can be due to a number of factors including the molecular structure and the type and number of hydroxyl groups, sugars and acids attached and the cellular environment such as pH or ultrastructure. Of the many anthocyanin pigments it is cyanidin, in the form of cyanidin 3-0-galactoside, which is primarily responsible for the red colouration in apple skin and the enzymes in this biosynthetic pathway for apple have been well described (Kim et al., 2003, Plant Science 165, 403-413; Honda et al., 2002, Plant Physiology and Biochemistry 40, 955-962). It has long been observed that anthocyanins are elevated in response to particular environmental, developmental and pathogenic stimuli. Research into apple fruit has demonstrated both the environmental and developmental regulation of anthocyanin accumulation. Pigment biosynthesis can be induced when fruit are subjected to white light, or more significantly, UV light, a phenomenon also observed in other species. Furthermore, anthocyanin levels can be elevated by cold temperature storage of the fruit. There is evidence for the coordinate induction of anthocyanin enzymes in a developmental manner in apple fruit with pronounced anthocyanin enzyme activity and correlated pigmentation increases in immature fruit and then again at ripening which appears to depend on the cultivar.
Studies show that there is highly specific regulation of genes in the anthocyanin pathway by specific binding of transcription factors (TFs) as complexes with promoter elements (Holton and Cornish, 1995, Plant Cell 7, 1071-1083). This regulation may also extend to non-pathway genes such as anthocyanin transport proteins.
MYB TFs have been shown to play an important role in transcriptional regulation of anthocyanins. Plant MYBs have been implicated in controlling pathways as diverse as secondary metabolism (including the anthocyanin pathway), development, signal transduction and disease resistance (Jin and Martin, 1999, Plant Mol Biol, 41, 577-585). They are characterised by a structurally conserved DNA binding domain consisting of single or multiple imperfect repeats; those associated with the anthocyanin pathway tend to the two-repeat (R2R3) class. Regulation can also be specific to discreet groups of genes, either early or late in the anthocyanin biosynthetic pathway. In the leaves of perilla, Perilla fruitescens, TF-driven regulation has been observed in virtually all stages of anthocyanin biosynthesis from CHS to the resultant anthocyanin protein transport genes whilst in grape, Vitis vinifera, specific regulation by MybA is restricted to the end-point of protein production (UFGT).
There are approximately 140 R3 MYB TFs in Arabidopsis, divided into 24 sub groups (Stracke et al. 2001, Current Opinion in Plant Biology, 4, 447-556). The Production of Anthocyanin Pigment 1 (PAP1) MYB (Borevitz et al., 2000, Plant Cell, 12, 2383-2394) falls into subgroup 10 (when the phylogeny of Stracke et al., 2001 is used) and demonstrates a high degree of amino acid conservation with other known anthocyanin regulators. When PAP1 was overexpressed in transgenic Arabidopsis this led to up-regulation of a number of genes in the anthocyanin biosynthesis pathway from PAL to CHS and DFR (Borevitz et al., 2000, Plant Cell, 12, 2383-2394; Tohge et al., 2005, Plant Journal, 42, 218-235).
In general MYBs interact closely with basic Helix Loop Helix TFs (bHLH), and this has been extensively studied in relation to the production of flavonoids (Mol et al., 1996; Winkel-Shirley, 2001). Examples include the maize ZmC MYB and ZmB bHLH and the petunia AN2 MYB and AN1/JAF13 bHLHs (Goff et al., 1992 Genes Dev, 6, 864-875; Mol et al., 1998, Trends in Plant Science, 3, 212-217). Evidently there is a degree of conservation, in different species, for this co-ordination. However, a MYB-bHLH partnership is not always necessary. Results from the overexpression of PAP1 suggested that, like the Maize P MYB (Grotewold et al., 2000 Proc Natl Acad Sci USA, 97, 13579-13584) and Arabidopsis MYB12 (Mehrtens et al., 2005 Plant Physiology, 138, 1083-1096), PAP1 did not require an over-expressed bHLH co-regulator to drive a massive increase in anthocyanin production. However, further studies showed that PAP1 does interact closely with bHLHs leading to stronger promoter (DFR) activation in in vivo assays (Zimmermann et al., 2004 Plant J, 40, 22-34). More recently, integrated transcriptome and metabolome analysis of PAP1 over-expressing lines confirmed PAP1 upregulates the bHLH TT8 (At4g09820) by 18-fold (Tohge et al., 2005, Plant J, 42, 218-235). This dependency on a co-regulator is linked to a small number of amino acid changes in the highly conserved R2R3 binding domain as evident in the comparison between the bHLH independent maize P and the bHLH dependent maize C1 MYBs, and is sufficient to direct activation of distinct sets of target genes (Grotewold et al., 2000, Proc Natl Acad Sci USA, 97, 13579-13584). In this study substitution of just six amino acids from the R2R3 domain of C1 into the corresponding positions in P1 resulted in a mutant with bHLH-dependent behaviour similar to C1. More recently it was suggested that this may be a key mechanism which permits MYBs to discriminate between target genes (Hernandez et al., 2004, J. Biol. CHem, 279, 48205-48213). These key amino acids are marked on FIG. 1. In contrast to PAP1, FaMYB1, represses anthocyanin biosynthesis during the late development of strawberry fruit. Despite this alternative role FaMYB1 shares homology with activation MYBs and can interact with (activation) bHLHs such as the Petunia AN1 and JAF13 (Aharoni et al., 2001, Plant J, 28, 319-332). Despite key residues being the same for PAP-like activators and FaMYB-like repressors, activators tend to fall in subgroup 10 while repressors fall in subgroup 17 (according to Stracke et al.).
An additional level of anthocyanin regulation involves a separate class of proteins, containing WD40 domains, which form complexes with MYB and bHLH proteins (as reviewed in Ramsay and Glover, 2005, Trends in Plant Science, 10, 63-70). Examples include an11 in petunia (de Vetten et al., 1997 Genes Dev, 11, 1422-1434) and TTG1 in Arabidopsis (Walker et al., 1999, Plant Cell, 11, 1337-1350). The transcriptional control of anthocyanins may be further complicated by tissue specific regulation (Kubo et al., 1999, Plant Cell, 11, 1217-1226) and possibly different layers of regulation dependent on stimuli such as cold, light and developmental cues (Davuluri et al., 2005, Nature Biotechnology, 23, 890-895).
Although studies into the activation and repression of anthocyanin synthesis in apple fruit have shown developmental and environmental regulation, to date transcription factors regulating anthocyanin synthesis have not been identified in this species or any other deciduous fruit. The control of anthocyanin accumulation in apple is a key question in understanding and manipulating fruit colour. Identification of the factors that exert this control provides tools for moderating the extent and distribution of anthocyanin-derived pigmentation in fruit tissue.
It is therefore an object of the invention to provide transcription factor sequences which regulate anthocyanin production in apple species and/or at least to provide the public with a useful choice.